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Relationships between intermediate filaments and cell-specific functions in renal cell lines derived from transgenic
1Institut National de la Santé et de la Recherche Médicale, Paris, France.
Abstract:
Four renal cell lines were derived from glomeruli, proximal, distal, and cortical collecting tubules microdissected from the kidneys of transgenic mice carrying the temperature-sensitive mutant of the simian virus 40 large T antigen under the control of the vimentin promoter. All four cell lines contained large T antigen in their nuclei, grew rapidly, and contained vimentin filaments when grown in serum-enriched medium at the permissive temperature of 33 degrees C. The glomerular cell line formed multiple layers of cells and contained smooth muscle actin and desmin filaments, features of mesangial cells. The three tubule cell lines formed monolayers of polarized cuboid cells separated by tight junctions and having a patchy distribution of cytokeratins K8-K18. A shift from 33 degrees C to the restrictive temperature (39.5 degrees C) stopped cell growth in all cell lines and caused profound changes in the content of intermediate filaments. Vimentin was still present in mesangial-like cells, but the proximal, distal, and collecting tubule cells contained uniform networks of cytokeratins K8-K18 and desmoplakin I and II around the cell peripheries. Potassium transport, mediated by Na+-K+ ATPase pumps and specific cAMP hormonal sensitivities, significantly increased in proximal, distal, and collecting tubule cells when shifted from 33 degrees C to 39.5 degrees C. Thus, the temperature-dependent inactivation of large T antigen, responsible for the arrest of cell growth, did not affect the phenotype of mesangial-like glomerular cells but induced some changes in the expression of intermediate filaments and restored, at least partially, the main parental cell-specific functions in proximal, distal, and collecting tubule cultured cells.
Insights
New mouse kidney cell lines, expressing simian virus 40 large T antigen, show temperature-dependent growth arrest. Tubule cells partially restore function, while glomerular cells maintain their phenotype.
Area of Science:
- Nephrology
- Cell Biology
- Molecular Biology
Background:
- Primary renal cell culture is crucial for studying kidney function and disease.
- Transgenic mouse models offer unique tools for cell line development.
- Understanding intermediate filament dynamics is key to cell differentiation and function.
Purpose of the Study:
- To establish and characterize novel renal cell lines from specific kidney compartments.
- To investigate the role of simian virus 40 large T antigen in regulating renal cell phenotype and function.
- To explore temperature-dependent changes in intermediate filament expression and cellular functions.
Main Methods:
- Microdissection of mouse kidney tubules and glomeruli.
- Establishment of transgenic mouse cell lines expressing temperature-sensitive simian virus 40 large T antigen.
- Cell culture at permissive (33°C) and restrictive (39.5°C) temperatures.
- Immunofluorescence microscopy for intermediate filament analysis.
- Assessment of ion transport and hormonal sensitivity.
Main Results:
- Four distinct renal cell lines (glomerular, proximal tubule, distal tubule, collecting tubule) were successfully derived.
- Temperature shift to 39.5°C arrested cell growth and altered intermediate filament expression.
- Glomerular cells maintained a mesangial-like phenotype, while tubule cells showed changes in cytokeratins and desmoplakin.
- Proximal, distal, and collecting tubule cells exhibited increased potassium transport and cAMP sensitivity at the restrictive temperature.
Conclusions:
- Temperature-sensitive simian virus 40 large T antigen effectively controls cell proliferation in these novel renal cell lines.
- Glomerular cell phenotype remains stable, while tubule cells partially regain parental cell-specific functions upon growth arrest.
- These cell lines provide valuable models for studying kidney development, differentiation, and function.